*Corresponding author. Email: B.Luke@cabi.org cCurrent address: c/o Mellingray Annex, Otterham Station, Cornwall, PL32 9YP, UK.

Using Particle Size Analysis to Determine the Hydrophobicity and Suspension of

Fungal Conidia with particular relevance to formulation of biopesticide

Belinda Lukea,b

*, Jane Faullb and Roy Bateman

a,c

aCABI, Bakeham Lane, Egham, Surrey TW20 9TY, UK Tel: 01491 829034 e:mail:

b.luke@cabi.org, roy@dropdata.org bBirkbeck College, Malet Street, London, WC1 7HX, UK Tel: 020 7631 6232 e-mail:

j.nicklin@bbk.ac.uk

mailto:b.luke@cabi.orgmailto:j.nicklin@bbk.ac.uk?subject=Email%20via%20department%20web%20site

B. Luke et al.

Abstract

Fungal formulations are vital for effective biopesticide development. Good

formulations help to optimise field efficacy while poor formulations result in product

failure. This study aimed to produce a hydrophobicity test that would be appropriate

for fungal conidia produced to a commercial quality and determine relative

hydrophobicity of fungi from four different genera by using laser diffraction. A

particle size analyser was used to determine the hydrophobicity of: three Metarhizium

acridum samples, M. anisopliae, Beauveria bassiana, Trichoderma stromaticum, T.

harzianum, T. viride and Alternaria eichhorniae conidia, by suspending the conidia in

three different liquids: Shellsol T (a mineral oil), water and 0.05 % Tween 80.

Hydrophobicity was determined by the size of the particles formed in each of the

liquids. All the Metarhizium samples were the most hydrophobic followed by B.

bassiana and A. eichhorniae. The Trichoderma samples were the least hydrophobic.

As a comparison a phase exclusion assay and a salt-mediated aggregation and

sedimentation (SAS) test were performed. It was not possible to get a reliable reading

for the B. bassiana, A. eichhorniae and T. viride samples using the phase exclusion

assay. The addition of salt in the SAS test did not affect the rate of sedimentation. It

was hypothesised that conidia size affected the results of the SAS test that made A.

eichhorniae the most hydrophobic conidia. Particle size analysis was a more accurate

test for comparing fungi from difference genera compared to the SAS test and phase

exclusion assay. PSA was also used to test three emulsions and demonstrated that

different formulations had an effect on particle size.

Keywords: formulation; microbial biopesticides; hydrophobicity; particle size

analysis; Metarhizium; Beauveria

B. Luke et al.

1. Introduction

When formulating fungal conidia it is crucial to understand how they disperse in

formulating media in order to maintain stability. Large particles, including

aggregations of fungal conidia are undesirable for two main reasons. Firstly if the

conidia are clumped the likely hit rate and hence control of the pest, whether the pest

is an insect, plant or fungus, is reduced due to uneven loading of the conidia in the

spray droplets (Bateman, 2004). Secondly large clumps in the formulation are more

likely to lead to blockages of the sprayer (Chapple et al., 2007).

There are numerous methods to determine the hydrophobicity of fungi including:

contact angle measurements, salt aggregation tests, phase distribution assays (Mozes

and Rouxhet, 1987), polystyrene microsphere assays (Clement et al., 1994) and salt

aggregation and sedimentation tests (Jeffs and Khachatourians, 1997); but depending

on the method used different results can be obtained (Mozes and Rouxhet, 1987). For

fungal conidia that are obviously at different ends of the hydrophobicity scale, most

methods can separate out the fungi into an order of the most hydrophilic to the most

hydrophobic. However, when dealing with fungi of different sizes and with similar

hydrophobicities it is sometimes hard to obtain a definitive rank. The rank of

hydrophobicity of a group of fungi may be an important factor as to which fungus is

chosen to formulate into a product (Talbot et al., 1996) or how much surfactant may

be required to obtain a homogenous suspension.

In this study a simple technique has been investigated to determine the relative

hydrophobicity of fungal conidia by using laser diffraction. A particle size analyser

was used to determine the relative hydrophobicity of fungal spores suspended in

B. Luke et al.

different polarity liquids. Not only does this method determine how easy it is for

conidia to be suspended in a liquid but also how the conidia, on its own or formulated,

interact with each other in a particular suspension. Two other hydrophobicity tests

were performed on the same fungal samples. A phase exclusion assay, where an

organic layer is added to an aqueous fungal suspension and the rate of migration of

the conidia into the aqueous phase is determined by using optical density (OD) as a

measure of the polar layer. The more hydrophobic the fungus the quicker it will

migrate into the non-aqueous layer and hence OD will decrease. Secondly, a salt-

mediated aggregation and sedimentation assay which also uses OD to measure the

rate at which conidia aggregate and sediment out of suspension (Jeffs and

Khachatourians, 1997). Those conidia that aggregate out fastest have a greater

hydrophobic nature than conidia left in suspension.

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2. Materials and methods

2.1. Fungal isolates

A range of fungi from four genera was used in this study (Table 1). Three different

batches of Metarhizium acridum, IMI 330189, were tested to see if there were any

differences in hydrophobicity of fungal conidia, when they were produced in different

ways. Of these, sample DM2 was produced in a laboratory and samples ME 006 and

ME 008 were produced by a commercial company. Beauveria bassiana and the

Trichoderma species were mass-produced by a two-stage process based on the

method used by Cherry, Jenkins, Heviefo, Bateman and Lomer (1999). The first

stage was a liquid culture followed by conidiation on a sterile solid substrate, Basmati

(Tilda) rice. However, Alternaria eichhorniae produced low conidial numbers when

mass produced on rice. Therefore, the second stage of mass production for A.

eichhoeniae was adapted by pouring the liquid culture onto foiled trays and allowing

the culture to dry slowly for the liquid to evaporate and conidiation to occur. The

fungi were harvested from the solid substrate using a ‘MycoHarvester v.1’ (Bateman,

2003, www.mycoharvester.info), which enables aerial conidia to be extracted from a

solid substrate while removing virtually all large fragments of mycelium or solid

substrate, so leaving mainly single conidia (Bateman et al., 2002). The

‘MycoHarvester’ was adapted for extraction of A. eichhorniae conidia by replacing

the substrate column (under negative pressure from the air intake at its base thus

creating a fluidized bed mechanism) with a suction tube to directly remove the

conidia from the trays. The conidia were dried to below 5 % moisture content by

placing the conidia in an airtight container with non-indicating silica gel beads for 5

days. Once the desired moisture content was achieved the conidia were packed in

hermetically sealed tri-laminate sachets and stored at 5 °C until required.

B. Luke et al.

2.2. Salt-mediated aggregation and sedimentation (SAS) test

This method was based on the one used by Jeffs and Khachatorians (1997). For each

fungal treatment, conidia were suspended in two buffer solutions; 2.0 mM di-sodium

hydrogen orthophospate buffer (pH. 6.8) and a 1:1 ratio of 2.0 mM di-sodium

hydrogen orthophospate buffer and 10 mM ammonium sulfate buffer. The resulting

conidial suspensions were vortexed for 10 seconds and their optical density (OD) was

measured using a spectrophotometer (Pharmacia, Pharmacia LKB, Novaspec II) set at

610 nm in 3.5 ml polystyrene cuvettes. Conidia were added until OD readings of 0.6

were achieved, the suspensions were incubated at 25 °C. After 30, 60 and 120

minutes the samples ODs were re-measured. The rate of sedimentation was

determined by calculating the percentage differences in OD between the original OD

reading and the subsequent readings. This experiment was replicated on three

separate occasions.

2.3. Phase exclusion assay

The phase exclusion assay was based on a method used by Mozes and Rouxhet

(1987). Conidia were suspended in 0.2 M tris buffer (pH. 7.0) and agitated in a vortex

blender for 10 seconds before measuring the OD at 610 nm. The suspensions were

adjusted for each sample to give an OD reading of 0.6. Once this was achieved 5 ml

toluene (anhydrous, 99.8%, Sigma-Aldrich) was added to each sample in a 1:1 ratio,

the samples were blended further for 20 seconds and incubated at 25 °C. After 30

minutes the aqueous layer was removed, being very careful not to remove any of the

toluene, and the OD of the aqueous layer was re-measured. The percentage of conidia

left in the aqueous layer was calculated. Those conidia that migrated to the organic

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layer at a faster rate were more hydrophobic than those spores left in the aqueous

layer. This experiment was replicated on three separate occasions.

2.4. Particle size analysis (PSA)

Conidia of each sample were suspended in 10 ml of three different liquids with

varying polarity: distilled water, 0.05 % Tween 80 in distilled water and Shellsol T

(Alcohols Ltd., Hertfordshire, UK) a paraffinic oil. Particle size spectra of the

resulting suspensions were measured with a Malvern 2600 particle size analyser

(Malvern Instruments Ltd., Spring Lane South, Malvern, Worcs., WR14 1AT, UK).

The instrument was fitted with a 63 mm lens using model independent analysis and a

PS1 sample cell that contained a small magnetic stirrer. Each reading consisted of a

background measurement with the blank formulating liquid, followed by the gradual

introduction of concentrated suspensions using a pipette. Each reading comprised of

1000 scans (equivalent to sub samples). Each sample was run twice through the

Malvern.

2.5. PSA of emulsion concentrates of IMI 330189

From the PSA testing M. acridum was shown to be very hydrophobic indicating that

these conidia prefer to be suspended in oil. However, a large majority of sprayers are

water-based. Hence M. acridum conidia were used to demonstrate the effects of

emulsion formulations in water on particle size. Commercially produced conidia of

IMI 330189 (see above) were prepared as emulsion concentrates using three different

emulsifiers (Table 2). First a stock suspension of conidia was prepared to mix with

the different emulsifiers in the following way:

Dry conidia 110 g/kg

B. Luke et al.

Structuring agent 10 g/kg

Ondina EL 880g/kg

The structuring agent was first dispersed in Ondina EL oil (a paraffinic oil) using a

Silverson L4RT mixer at approximately 6000 rpm for 2 minutes (Silverson Machines

Ltd., Chesham, Bucks, UK). This was followed by the addition of the dry conidia.

The conidia were mixed into the formulation for two minutes at approximately 6000

rpm.

The conidia stock suspension and the various emulsifiers were combined at the 5 %

level to make 3 x 250 g blends (Table 2). The resulting conidia emulsion concentrate

(EC) suspensions were mixed using the Silverson L4RT mixer for no more than two

minutes at approximately 6000 rpm. BotaniGard® ES, an emulsifiable suspension

mycoinsecticide (active ingredient Beauveria bassiana) used to control whitefly,

aphids, thrips and mealybugs in ornamentals and vegetables, was used as a standard

control to compare against the physical properties of the new formulations.

Emulsion particle/oil droplet size was measured with a Malvern 2600 particle size

analyser used in the same way as mentioned above. Each reading consisted of a

background measurement of distilled water followed by the gradual introduction of

emulsion concentrate using a pipette. A reading was taken when the obscuration of

the laser was optimal in the ‘illustrate live’ command. Measurements were repeated

to check for consistency and are presented here as means.

2.6. Statistics

All percentage data was arcsine transformed prior to any statistical tests being carried

out. To determine if the type of buffer had a significant effect on the rate of

B. Luke et al.

sedimentation, a one-way ANOVA was performed. Kruskal-Wallis non-parametric

tests were carried out on the transformed data to determine if the fungal isolate had an

effect on the rate of sedimentation, phase exclusion or particle size. All statistical

tests were carried out using SPSS for Windows version 17.0.0. to the 95%

significance level.

B. Luke et al.

3. Results

3.1. Salt-mediated aggregation and sedimentation (SAS) test

The SAS test is based on the principle that the more hydrophobic conidia will

aggregate together and hence sediment out of suspension at a faster rate than

those conidia which are more hydrophilic. The addition of salt had no

significant (P>0.05) effect on the sedimentation of any of the samples tested.

Thus data for the buffer and buffer and salt samples were amalgamated. There

were very highly significant differences (Chi-squared = 64.3; df = 8; p < 0.001)

in the rate of sedimentation of conidia (Figure 1). The Trichoderma isolates

were rated as the least hydrophobic fungal conidia with the least amount of

sedimentation. The Metarhizium and Beauveria isolates were in the next group

of fungi ranging from 53-71 % conidia left in suspension after 120 minutes. The

Alternaria isolate was ranked the most hydrophobic conidia when using the SAS

method with only 46 % of conidia left in suspension after 120 minutes.

3.2. Phase exclusion assay

The phase exclusion assay works on the principle that more hydophobic conidia will

migrate from an aqueous phase to a solvent phase at a faster rate than those fungi

which are more hydrophilic. Figure 2 shows the percentage of conidia left in the

aqueous phase after 30 minutes of being combined with toluene. Highly significant

differences showed that the Metarhizium samples were rated as the most hydrophobic,

followed by the Trichoderma sample FA 64 and the DIS 219f Trichoderma sample

was ranked as the least hydrophobic (Chi-squared = 15.251; df = 5; p = 0.009).

Practical difficulties with the B. bassiana, SP2 002, A. eichhorniae, WH3a and

Trichoderma sp., T22 samples resulted in large standard errors. Each time the

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experiment was replicated a different percentage of conidia left in aqueous suspension

was achieved, varying from 50 % to 250 % for SP2 002, 26 % to 193 % for WH3a

and 17 % to 102 % for T22. The number of conidia in the samples did not increase,

so the increase in OD was due to another, undetermined factor. For this reason these

results were omitted from the graph and statistical tests.

3.3. Particle size analysis (PSA)

PSA examined how conidia clumped or dispersed when suspended in liquids with

different polar properties. Hydrophilic conidia suspend better in water as singular

conidium giving a smaller size in the PSA test i.e. less clumping of conidia. Whereas,

hydrophobic conidia are more likely to suspend singly in Shellsol T giving a smaller

particle size under these test conditions. M. anisopliae isolate TNS 10 (Figure 3i), M.

acridum isolates ME 006 (Figure 3ii) and ME 008 (Figure 3iii), B. bassiana isolate

SP2 002 (Figure 3iv) and A. eichhorniae isolate WH3a (Figure 3v) all suspended

better in Shellsol T than water. When this is plotted on a graph, the curve for the

Shellsol T sample is relatively near the Y axis and relatively steep. For example, on

Figure 3i. 90 % of all particles were less than 10 µm in size when suspended in

Shellsol T. In contrast, the curves for the samples suspended in water are further

away from the Y axis and less steep. For example, on Figure 3i. 90 % of the particles

suspended in water were up to 95 µm in size. Isolates ME 006 and ME 008

suspended equally well in 0.05 % Tween 80 as compared to Shellsol T but, when

examined under a microscope, differences could be observed (Figure 4). Conidia

suspended in Shellsol T mainly showed single conidium evenly dispersed (Figure 4.i),

whereas conidia suspended in 0.05 % Tween 80 showed single conidium interspersed

with a few clumps (Figure 4.ii). The conidia suspended in only water showed no

B. Luke et al.

individual conidium but many clumps of varying sizes (Figure 4.iii). B. bassiana,

SP2 002 initially suspended very well in 0.05 % Tween 80 but, after 60 % of the

particles were suspended, particle size increased and at 90 % particle size was up to

49 µm compared to 21 µm when suspended in Shellsol T.

Three of the Trichoderma isolates, DIS 219f (Figure 5i), FA64 plate 0 (Figure 5ii) and

FA64 plate 5 (Figure 5iii) suspended better in water than in Shellsol T, suggesting

that these conidia are more hydrophilic than ME 006, ME 008, TNS 10, SP2 002 and

WH3a. When examined under a microscope DIS 219f (Figure 6) showed the opposite

of the M. acridum sample (Figure 4). There was clumping of conidia when suspended

in Shellsol T (Figure 6.i) but none when suspended in 0.05 % Tween 80 (Figure 6.ii)

or water (Figure 6.iii). The fourth Trichoderma isolate T22 behaved in a very similar

manner, regardless of suspending liquid, until around 50 – 60 % cumulative particles

were suspended, when 0.05 % Tween 80 had the smallest particle size, followed by

Shellsol T and then water (Figure 7).

M. acridum isolate DM 2 had very similar results for suspension in water and in

Shellsol T. The conidia suspended better in 0.05 % Tween 80 (Figure 8). However,

care has to be taken when interpreting these results as there were difficulties in

suspending conidia in the water phase as most of the conidia floated on the surface,

consequently unusual results occurred and this may account for the difference

between DM 2 and the other Metarhizium isolates.

3.4. PSA of emulsion concentrates of IMI 330189

B. Luke et al.

The PSA method was used to determine the effect of emulsion formulation on

particle/droplet size. The particle/droplet size of BotaniGard, the standard, was 14

µm. Formulations 1 and 2 were similar in particle size with a mean particle size of 22

µm and 15 µm, respectively. However, formulation 3 had a mean particle/droplet size

of 43 µm. That is over 300 % larger than BotaniGard.

B. Luke et al.

4. Discussion

The study of hydrophobicity of fungal conidia can be approached using many

different methods but none seem to give a consistent answer. A summary of the

rankings of hydrophobicity for the phase exclusion assay, the SAS test and the PSA

method are shown in Table 3. Of the three tests the PSA rankings were the most

accurate when samples were checked by microscopic examination of the conidial

suspension in the different suspending liquids (Figure 4). The SAS test was the next

most reliable test, excluding Alternaria due to size differences. The phase exclusion

test did not give reliable results for the Beauveria and Alternaria samples.

The conidia used in this study were mass produced using a two phase process which

produced very hydrophobic conidia. The conidia were dried to a moisture content

(MC) of approximately 5 %. This is known to be a suitable moisture content for

storage of certain fungi such as Metarhizium and Beauveria (Hong et al., 1997; Hong

et al., 2000; Hong et al., 2002). The fungi used by other researchers were probably

not dried to a MC of 5 % and in most cases the fungal spores were stored in a liquid

suspension (Jeffs et al., 1999; Jeffs and Khachatourians, 1997; Mozes and Rouxhet,

1987). Shan et al. (2010) produce and dried conidia in a similar manner to the current

study, i.e. aerial conidia dried to 5 % MC. To overcome problems in assessing

conidial hydrophobicity, using an aqueous-solvent partitioning method, they added

0.02 % Tween 80 to help suspend the conidia. However, no mention was made of the

possible effect of Tween 80 on the hydrophobicity on conidia.

The results from the emulsion particle/oil droplet size indicate that different

formulating oils and emulsifiers can give very different particle sizes when suspended

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in water. Droplet size is directly proportional to its size (Gan-Mor and Matthews,

2003). This means that relatively fewer, larger, emulsion droplets will have a higher

number of conidia present in them compared to smaller emulsion droplets. When

sprayed the larger emulsion droplets will give a less even coverage as the conidia are

clumped into relatively fewer droplets compared to the smaller emulsion droplets

resulting in decreased efficacy of the application (Gan-Mor and Matthews, 2003).

This study highlighted the importance of checking formulations as formulation 3 had

a very high mean particle size compared to the standard control and other

formulations. Ettmueller et al. (1995) concluded similar findings when they

evaluated the distribution and sedimentation of dispersion chemical formulations,

such as suspension concentrates, in spray tanks and found that different emulsifiers

suspended and resuspended with different ‘ease’.

For operational reasons, high quality formulations consisting of stable suspensions,

require stringent particle size specifications (i.e. consisting mostly of single conidia

with mycopesticides) that would usually be monitored with a PSA (Cherry et al.,

1999). Use of such instruments is therefore practical and a good method of not only

determining hydrophobicity of the conidia but also of examining how conidia interact

within a particular liquid. Conidia may suspend in a liquid but within that suspension

the conidia may be clumped and/or unevenly distributed (Bateman, 2004). The PSA

method not only indicated which was the more suitable liquid but also allowed

clumping to be detected. This is vitally important for formulation of conidia as

formulations need to be homogeneous to ensure a stable formulation, an even spray,

and hence a greater hit rate after application (Burges, 1998).

B. Luke et al.

The difficulties encountered when some conidia did not suspend easily, in the PSA

method, i.e. B. bassiana in water, resulted in ‘false’ readings. Only a subsample of

the conidia, those that were relatively hydrophilic compared to the majority, were

recorded. An improvement in the method would have been to rank samples that did

not suspend well in Shellsol T, or water as either highly hydrophilic or highly

hydrophobic, respectively.

The SAS method ranked A. eichhorniae, isolate WH3a, as the most hydrophobic

sample, in contrast to the other two tests, which ranked A. eichhorniae as the second

least hydrophobic fungus. These contrasting results may be due to the size of A.

eichhorniae conidia which can vary in size from 20 to 69 µm depending on the age of

the conidia (David 1991), compared to less than 10 µm for the other fungal conidia

examined in this study (Kirk et al., 2008). As A. eichhorniae conidia are up to an

order of magnitude greater in size than the other conidial samples, in compliance with

Stokes’ law, the conidia are going to settle at a faster rate. Hence, some caution has to

be taken when using the SAS method to rank the hydrophobicity of different sized

fungal conidia.

In the phase exclusion assay B. bassiana, SP2 002, A. eichhorniae, WH3a and

Trichoderma sp., T22, conidia, when mixed with the toluene phase, did not separate

out fully after 30 minutes. This was most evident in the B. bassiana sample, with

small bubbles of the aqueous phase captured in the toluene layer. Hence, it was very

difficult to get enough of the aqueous layer to take an accurate reading. The strong

interactions of the conidia with the water interfered with the OD readings. There

were also some instances where the OD reading for B. bassiana, A. eichhoriae and

B. Luke et al.

Trichoderma sp. conidia actually increased. Two possible explanations for this is that

firstly conidia had an emulsifier affect when the water and toluene layers were mixed

causing the layers to form an emulsion of sorts. Secondly conidia may have imbibed

water and increased in size. Imbibition would result in larger particles being detected

and less light able to pass through the sample, resulting in an OD reading greater than

the original reading. However, 30 minutes was probably not long enough for the

conidia to imbibe sufficient water to swell up. With work on Aspergillus fumigatus,

Renwick et al. (2006) demonstrated that it took 2 hours before conidia were observed

to be swollen. Further studies would need to be carried out to determine why the OD

reading increased.

The biggest limitation encountered with all the methods was suspending very

hydrophobic conidia in an aqueous liquid. This resulted in difficulty in reading

particle size as the conidia floated on surface of the water. For the phase-exclusion

assay and the SAS tests it was difficult to get an initial OD reading of 0.6 as conidia

would float on the surface of the water. This problem does not seem to be mentioned

in other studies on hydrophobicity (Jeffs and Khachatourians, 1997; Mozes and

Rouxhet, 1987). Some conidia will suspend into an aqueous suspension as each

conidium is slightly different and hence hydrophobicity will vary between conidia and

between isolates of the same species. Thus while a small percentage of the relatively

less hydrophobic conidia are suspended in the aqueous phase, the majority of the

conidia will be floating on the surface of the liquid or stuck to the side of the

container (personal observation). This is not a true reading but a small sub-sample of

the population.

B. Luke et al.

In conclusion, the PSA method was a quick and simple way to test the relative

hydrophobicity of fungal conidia. Size of the conidia did not affect the results as

encountered when using the SAS method and no extraction was required as in the

phase exclusion assay, where difficulties occurred. In addition to the PSA method

determining relative hydrophobicity, it also helped to explain how the conidia react

with each other, i.e. clumping, when suspended in different liquids, and how non-inert

formulation ingredients affected particle size, which would be of interest to a

formulation scientist.

B. Luke et al.

Acknowledgements:

We gratefully acknowledge support from CABI and thank Dave Moore and Steve Edgington

for critical review of the manuscript.

B. Luke et al.

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Legends:

Table 1. Fungi used in this study. The isolate number represents conidia stored in the

CABI genetic resource collection (commonly known as the IMI collection), which is

part of the UK culture collection. The code represents identification codes for this

experiment. * = not all isolates were logged in the CABI collection as they were held

locally.

Table 2. Emulsifier type and quantity used in the formulation.

Table 3 Comparison of the ranks of hydrophobicity obtained for the different fungal

groups using three different hydrophobicity determining methods. PE = phase exclusion, SAS = salt and sedimentation assay and PSA = particle size analysis. Rank

is 1= the most hydrophobic and 4 = the least hydrophobic.

Figure 1. Percentage of conidia left in suspension after 120 minutes of being mixed

with buffer solutions, using the salt-mediated aggregation and sedimentation method.

Error bars = standard error.

Figure 2. Percentage of conidia in the aqueous phase after 30 minutes of being

combined with toluene using the phase exclusion assay. Error bars = standard error.

Figure 3. Cumulative particle size of different fungi suspended in three different

liquids. Key: ♦ = suspended in water, ▲ = suspended in Shellol T and ■ = suspended

in 0.05 % Tween 80.

Figure 4. Microscopic examination of M. acridum, IMI 330189, conidia suspended in

Shellsol T (i), 0.05 % Tween 80 (ii) and water (iii). Photograph by Roberto Alves.

Figure 5. Cumulative particle size of Trichoderma sp. (three different samples, i. T.

harzianum, DIS 219f, ii. T. stromaticum plate 0 and iii. T. stromaticum plate 5)

suspended in three different liquids. Key: ♦ = suspended in water, ▲ = suspended in

Shellol T and ■ = suspended in 0.05 % Tween 80.

Figure 6. Microscopic examination of T. harzianum, DIS 219f, conidia suspended in

Shellsol T (i), 0.05 % Tween 80 (ii) and water (iii). Photograph by Roberto Alves.

Figure 7. Cumulative particle size of Trichoderma viride isolate T22 suspended in

three different liquids. Key: ♦ = suspended in water, ▲ = suspended in Shellol T and

■ = suspended in 0.05 % Tween 80.

Figure 8. Cumulative particle size of M. acridum suspended in three different liquids.

Key: ♦ = suspended in water, ▲ = suspended in Shellol T and ■ = suspended in 0.05

% Tween 80.

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Table 1.

Fungus Isolate No. Code

Metarhizium acridum IMI 330189 DM2

Metarhizium acridum IMI 330189 ME 006

Metarhizium acridum IMI 330189 ME 008

Metarhizium anisopliae IMI 385045 TNS 10

Beauveria bassiana IMI 390162 SP2 002

Trichoderma stromaticum * FA 64

Trichoderma harzianum IMI 385767 DIS 219f

Trichoderma viride * T22

Alternaria eichhorniae * WH3a

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Table 2.

Formulation Emulsifier type Percentage of emulsier in

formulation

1 Non-ionic 5 %

2 2 parts: a. Anionic/nonionic blend

b. non-ionic

a. 2.8 % and b. 2.2 %

3 Anionic/nonionic blend 5 %

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Table 3.

Fungus Rank

PE SAS PSA

Metarhizium sp. 1 3 1

Beauveria 4 2 2

Alternaria 3 1 3

Trichoderma sp. 2 4 4

.

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Figure 1.

Trichoderma spp.

Metarhizium and Beauveria spp.

Alternaria sp.

Least Hydrophobic Most Hydrophobic

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Figure 2

Metarhizium spp.

Trichoderma spp.

Most hydrophobic Least hydrophobic

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i. M. anisopliae TNS 10

ii. M. acridum ME 006

iii. M. acridum ME 008

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iv. B. bassiana SP2 002

v. A. eichhorniae WH3a

Figure 3.

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Figure 4.

i

ii

iii

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i. Dis 219f

ii. FA64 plate 0

iii. FA64 Plate 5

Figure 5.

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Figure 6.

i

ii

iii

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Figure 7.

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Figure 8.